Methods for analyzing PRLTS DNA

ABSTRACT

A gene is provided which is present in the deletion region of a chromosome common in lung cancer, hepatocellular carcinoma and colorectal cancer and encodes a novel protein, a protein encoded by the gene (PRLTS protein), and a method of discriminating tumor cells.

This is a divisional of Ser. No. 08/506,864, now U.S. Pat. No.5,834,245, filed Jul. 25, 1995.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to PRLTS proteins, DNAs encoding theproteins and methods of discriminating tumor cells in which use is madeof the DNAs. The present invention is usefully applied in the fields ofmedical science and pharmaceuticals.

2. Description of the Related Art

There has long been a conception that mutations in cellular proteinsplay an important role in carcinogenesis. The progress of geneticengineering achieved in recent years has made it possible to analyze theamplification of the DNA encoding a specified protein and gene mutationsin tumor cells, thereby rapidly advancing cancer research.

The analysis and identification of genes (oncogenes) encoding proteinsbelieved as participating in the malignant alteration of cells and theabnormal proliferation of tumor cells have been promoted, so that theidentification of such genes number in the tens. On the other hand,counteracting genes (tumor suppressor genes) are highlighted in recentyears. Tumor suppressor genes hitherto discovered include the Rb genecapable of suppressing retinoblastoma Friend, S. H., et al., Proc. Natl.Acad. Sci. USA., 84, 9095 (1987)!, the p53 gene capable of suppressingcolorectal cancer Lane, D. P., et al., Nature, 278, 261 (1979)!, the APCgene capable of suppressing colorectal cancer Kenneth, W. K., et al.,Science, 253, 661 (1991)! and the WTI gene capable of suppressing Wilms'tumor Call, K. M., et al., Cell, 60, 509 (1990)!. With respect to thep53 gene, cases are known in which germ-line mutations in the gene areinherited "Li-Fraumeni syndrome" (Makin, D., et al., Science, 250, 1233(1990); and Srivastava, S., et al., Nature, 348, 747 (1990))!. It isgradually becoming apparent that defects in not only a single gene butalso in multiple genes participate in the progression of the malignantphenotype of cancer, and it is believed that there will be furtherdiscovered a large number of unidentified oncogenes and tumor suppressorgenes. Their discovery and elucidation are anticipated by not onlyresearch and clinical experts but also people worldwide.

It is estimated that there are about 4500 genes on the human chromosome8, and to now the presence of genes causative of genetic diseases suchas Langer-Gideorn syndrome, Werner syndrome and pigmentous retinitis issuggested on this chromosome. The present inventors and otherresearchers reported Emi, M., et al., Cancer Res., 52, 5368-5372 (1992)!that on the short arm of the human chromosome 8 there is a sitefrequently deleted in various tumors such as lung cancer Ohata, H., etal., Genes Chromosomes & Cancer, 7, 85-88 (1993)!, hepatocellularcarcinoma Emi, M., et al., Genes Chromosomes & Cancer, 7, 152-157(1993)!, colorectal cancer Fujiwara, Y., et al., Cancer Res., 53,1172-1174 (1993)!, prostatic cancer Bererheim, U., et al., GenesChromosomes & Cancer, 3, 215-220 (1991)! and bladder cancer (Knowles, M.A., et al, Oncogene, 8, 1357-1364 (1993)! and that, hence, the presenceof an important tumor suppressor gene capable of participating invarious cancers is foreseen in the above deletion site.

Therefore, isolation of the causative gene present in the above site andidentification of a protein corresponding thereto are now themes ofgreat concern of not only worldwide doctors and researchers but also thegeneral public, whose attainment is highly anticipated.

DISCLOSURE OF THE INVENTION

Summary of the Invention

An object of the present invention is to provide a novel protein relatedto tumors such as lung cancer, hepatocellular carcinoma, colorectalcancer and prostatic cancer, a gene encoding the same, and a method ofdiscriminating tumor cells from normal cells with the use thereof.

The present inventors have further localized the region of the short armof the human chromosome 8 which is commonly deleted in lung cancer,hepatocellular carcinoma and colorectal cancer.

More specifically, numerous cosmid clones having DNA fragments of thehuman chromosome 8 introduced thereinto have been prepared, and theirpositions on the chromosome has been determined in accordance with thefluorescent in situ hybridization technique FISH technique; Inazawa etal., Genomics, 10, 1075-1078 (1991)!. From among the resultant markers,one having the property of having different restriction fragment lengthsdepending on the individual (RFLP: restriction fragment lengthpolymorphism), i.e., an RFLP marker has been selected.

The RFLP marker has the feature that, by the use thereof, two homologouschromosomes inherited from the parents can be discriminated according tothe difference in polymorphism when, however, both exhibit the samepolymorphic pattern, the discrimination cannot be made (notinformative)!. Such a phenomenon that this difference in polymorphicpattern between the two homologous chromosomes (heterozygosity) ispresent in normal tissues and disappears in carcinoma tissues (LOH: lossof heterozygosity) means the deletion of the RFLP marker site in one ofthe homologous chromosomes. Further, generally, it is believed that theinactivation of tumor suppressor gene on both of the chromosomes whichis attributed, for example, to a deletion in one of a pair ofchromosomes and a mutation in the other leads to a malignant alterationand that a tumor suppressor gene is present in a region commonly deletedin numerous tumors.

The present inventors have investigated the LOH of the short arm of thechromosome 8 with respect to each of about 100 cases of humanhepatocellular carcinoma, about 120 cases of colorectal cancer and about50 cases of nonsmall cell lung cancer by the use of the obtaineddetailed physical chromosomal map and the RFLP marker. As a result, theminimal deletion region which is common in them has been identified.

Partial deletions in the short arm of the chromosome 8 in humanhepatocellular carcinoma (HCC) and lung cancer (LC) are shown in FIG. 1.Each solid circle refers to the loss of heterozygosity (LOH), while eachopen circle to the retention thereof.

Partial deletions in the short arm of the chromosome 8 in colorectalcancer (CRC) are shown in FIG. 2. Each solid circle refers to the lossof heterozygosity (LOH), while each open circle to the retentionthereof.

The designation of each cosmid clone used as a probe, the determinedposition thereof on the chromosome, and the LOH are collectively givenin Table 1.

                                      TABLE 1    __________________________________________________________________________                             loss of hetero-    designa-         physical  probe  allelle                               zygosity (LOH)    tion of         localiza- fragment                          size HCC                                  CRC                                     LC    probe         tion  enzyme                   (kb)   (kb) (%)                                  (%)                                     (%)    __________________________________________________________________________    C18-1344         8p22  Msp1                   Msp1   4.5  10/40                                  25/71                                     8/30                   4.8    2.6/1.9                               (25)                                  (35)                                     (27)    C18-2195         8p21.3-p22               Msp1                   Taq 1  3.6  7/32                                  15/35                                     6/20                   5.0    2.6  (22)                                  (43)                                     (30)    C18-2014         8p21.3-p22               Taq1                   Taq 1  3.9  2/6                                  7/24                                     7/17                   3.9    2.2/1.7                               (33)                                  (29)                                     (41)    cMSR-32         8p22 (MSR)               Msp1                   E-H    6.3/3.1                               10/54                                  27/74                                     16/38                   0.9    /2.9/2./                               (19)                                  (36)                                     (42)                          (4 alleles)    cMSR-35         8p22 (MSR)               Taq1                   Tsq 1  2.4  3/9                                  16/33                                     12/20                   2.4    1.6  (33)                                  (48)                                     (60)    C18-2644         8p21.3-p22               Msp1                   Msp 1  5.5                   2.9    2.9  2/6                                  9/21                                     15/22               Taq1                   Msp 1  4.0  (33)                                  (43)                                     (68)                   2.4    0.6    C18-1051         8p22-p21.3               Msp1                   Msp 1  4.3  10/33                                  19/42                                     10/21                   4.3    4.0  (30)                                  (45)                                     (48)    C18-487         8p22-p21.3               Msp1                   Msp 1  1.2  5/14                                  10/21                                     3/7         (D85233)  1.2    1.0  (36)                                  (48)                                     (43)    C18-245         8p22-p21.3               Taq1                   Msp 1  4.8  4/18                                  15/30                                     12/27         (D85335)  7.0    4.2 and 3.0                               (22)                                  (50)                                     (44)                          1.8/1.2    C18-2439         8p21.3-p22               Taq1                   Msp 1  2.3  9/39                                  14/48                                     7/22                   2.8    1.6  (23)                                  (29)                                     (32)    C18-1312         8p21  Taq1                   Msp 1  2.0  9/40                                  22/49                                     7/23                   2.8    1.5  (23)                                  (45)                                     (30)    C18-190         8p21.3               Taq1                   Taq 1  4.8  8/37                                  18/49                                     8/19         (D85334)  3.5    3.5  (22)                                  (37)                                     (42)    C18-1013         8p21.3               Msp1                   Msp 1  3.5  16/54                                  31/51                                     5/24                   2.7    2.7  (30)                                  (61)                                     (21)    C18-1308         8p21  Msp1                   Msp 1  4.6  7/25                                  8/20                                     4/17                   4.6    3.5  (28)                                  (40)                                     (24)    __________________________________________________________________________

The above regions conform to each other, and two YAC (Y738 and Y812)clones covering the most localized common deletion region (8p21.3 to8p22) have been isolated (see FIG. 3).

A cosmid contig covering the common deletion region has been constructedby subcloning the YAC DNA into cosmids.

DNA fragments capable of serving as exons have been taken throughselection from the above cosmids according to an exon amplificationtechnique. Subsequently, cDNA libraries have been screened with the useof the above DNA fragments as probes, thereby obtaining a plurality ofcDNA clones derived from the genes present in the common deletionregion. Whether or not a tumor-tissue-specific gene rearrangement isdetected has been investigated with the use of these cDNA clones asprobes according to the Southern blotting analysis. As a result, whenone cDNA clone has been used as a probe, a gene rearrangement apparentin one case of pulmonary nonsmall cell carcinoma has been recognized.

This clone has encoded a novel protein exhibiting a significantsimilarity to the N-terminal domain (extracellular domain) of each ofplatelet-derived growth factor receptor b PDGFR-b; Yarden, Y., et al.,Nature, 323, 226-232 (1986)! and fms-like tyrosine kinase flt; Shibuya,M., et al., Oncogene, 5, 519-524 (1990)!. This protein has beendesignated PRLTS (PDGF receptor beta-like tumor suppressor) protein.Whether or not there are any mutations in human hepatocellularcarcinoma, pulmonary nonsmall cell carcinoma and colorectal cancer withrespect to the nucleic acid sequence of the coding region of this genehave been investigated. As a result, three cases of apparent mutationsin the gene have been identified. Thus, it has become apparent that thedefect or deficiency in this protein and the deletion or mutation in theDNA encoding the protein deeply participate in the progression ofvarious tumors including hepatocellular carcinoma and pulmonary nonsmallcell carcinoma.

The present invention is of the utmost importance in that, throughinvestigations of, for example, whether or not there is any defect ordeficiency in this protein and whether or not there is any deletion ormutation in the DNA encoding the protein, it provides schemes andmaterial for resolving difficult problems such as risk diagnosis, earlydetection, progress observation, determination of treatment plan andpresumption of prognosis at least as regards hepatocellular carcinoma,pulmonary nonsmall cell carcinoma and colorectal cancer, so that thisfield of technology can be rapidly advanced.

Thus, the present invention relates to (1) a PRLTS protein having anamino acid sequence comprising the whole or a part of the amino acidsequence specified in sequence ID NO 1, (2) a DNA having a nucleic acidsequence comprising the whole or a part of the nucleic acid sequencespecified in any of sequence ID NO 2, 3, 4, 5, 6, 7, 8 and 9, (3) avector containing a DNA having a nucleic acid sequence comprising thewhole or a part of the nucleic acid sequence specified in sequence ID NO2, (4) a transformant having, introduced thereinto, the vector describedin the above item (3), (5) a process for producing the PRLTS proteindescribed in the above item (1) which comprises using the transformantdescribed in the above item (4), (6) a DNA probe or primer (which may bea single stranded one) having a sequence comprising the whole or a partof the nucleic acid sequence specified in sequence ID NO 2, (7) anantiserum, a polyclonal antibody or a monoclonal antibody capable ofcombining with a PRLTS protein having the amino acid sequence specifiedin sequence ID NO 1, and (8) a method of discriminating tumor cellswhich comprises detecting the genomic mutation of a DNA encoding a PRLTSprotein having an amino acid sequence comprising the whole or a part ofthe amino acid sequence specified in sequence ID NO 1.

The present invention comprehends proteins which are substantiallyequivalent to the above protein and which are obtained by addition,deletion, insertion or substitution of one or more constituent aminoacids of the above protein, and DNAs which are substantially equivalentto the above DNA and which are obtained by addition, deletion, insertionor substitution of one or more constituent bases (nucleotides) of theabove DNA.

The DNA having a nucleic acid sequence comprising the whole or a part ofthe nucleic acid sequence of the DNA according to the present invention,or comprising a sequence complementary to the whole or a part of thenucleic acid sequence of the DNA according to the present invention, canbe utilized in gene analysis and diagnosis as the primer or probe. Theterm "part of the nucleic acid sequence" as used herein means a sequenceof at least six continuous bases (nucleotides), preferably at leasteight bases, and still more preferably 10 to 12 bases or 15 to 25 basescorresponding to (i.e., contained in or complementary to) the nucleicacid sequence of the DNA encoding the PRLTS protein. The primer or probeof the present invention, which is an oligonucleotide or polynucleotide,may also contain at least one base not corresponding to the nucleic acidsequence of the DNA encoding the PRLTS protein.

The whole or a part of the PRLTS protein can be used as an epitope inthe preparation of antibodies and as agents for research and diagnosisusing such antibodies. The term "epitope" as used herein refers to anantigenic determinant of a polypeptide. It is publicly known that apolypeptide composed of 6 amino acids combines with an antibody see WOof PCT Patent Applications No. 8403564, published on Sep. 13, 1984(Assignee: COMMONWEALTH SERUM LABS AND GEYSEN, H. M.)!. The term "partof the amino acid sequence" as used herein means a sequence of at leastsix continuous amino acids, preferably at least eight to ten aminoacids, and still more preferably at least 11 to 20 amino acidscorresponding to (i.e., contained in) the amino acid sequence of thePRLTS protein according to the present invention. The polypeptide havingan amino acid sequence comprising a part of the amino acid sequencedescribed above may also contain at least one amino acid notcorresponding to the amino acid sequence of the PRLTS protein.Naturally, polypeptides each composed of at least 20 consecutive aminoacids can also be used.

Detailed Description of the Invention

The present invention will now be described in greater detail.

(1) Isolation of cDNA clone

Cosmid clones of the human chromosome 8 can be prepared in, for example,the following manner. From a human-mouse hybrid cell line containing asingle human chromosome 8 in a mouse genomic background, a genomic DNAis extracted. Then, DNA fragments of the genomic DNA can be integratedinto a vector, such as pWEX15, according to the method reported byTokino et al. Tokino et al., Am. J. Hum. Genet., 48, 258-268 (1991)!.Then, clones having an insert originating from the human chromosome canbe screened therefrom by conducting colony hybridization with the use ofa whole human DNA as the probe.

The thus-obtained large number of cosmid clones containing a DNAoriginating from the human chromosome 8 are then subjected to the FISHtechnique. Thus, each of the large number of cosmid clones can belocalized throughout the chromosome, and a detailed physical chromosomalmap can be prepared with the use of the cosmid clones as the marker.Further, RFLP markers can be isolated on the basis of the fragmentlength pattern of which has been prepared by the digestion of human DNAwith several restriction enzymes Nakamura et al., Am. J. Hum. Genet.,43, 854-859 (1988)!. With the use of the above map and RFLP markers, theDNA of a tumor tissue of a patient is examined in the LOH (loss ofheterozygosity). Thus, the common deletion region on the chromosome inthe tumor tissue can be localized into an extremely restricted region inthe vicinity of from p21.3 to 22 of the human chromosome 8.

A cosmid contig of this localized common deletion region can beconstructed by selecting a YAC clone having the genomic DNA of thisregion and subsequently preparing a cosmid library on the basis of theDNA of the YAC clone. Further, DNA fragments having sequences capable ofserving as exons can be selected from restriction enzyme fragments ofthe cosmid clones according to the exon amplification technique Buckleret al., Proc. Natl. Acad. Sci. USA., 88, 4005-4009 (1991)!. cDNAs of thegenes present in the localized region in the vicinity of from p21.3 to22 of the human chromosome 8 can be cloned by screening cDNA librarieswith the use of the thus-obtained DNA fragments as probes. A clonehaving a sequence included in the tumor-tissue-specific generearrangement can be screened from the above cDNAs according to theSouthern blotting analyses. The nucleic acid sequence of the cDNA can bedetermined according to the common procedure (Maniatis, J., et al.,Molecular Cloning 2nd. ed., Cold Spring Harbor Laboratory Press, N.Y.,1989).

The cDNA clone thus-obtained can be confirmed to be the desiredcausative gene clone by searching for the presence of mutation and thefrequency of mutation in cancer patients according to the SSCP methodOrita, M., et al., Genomics, 5, 874-879 (1984) and Orita, M., et al.,Cell, 60, 509-520 (1990)! or the RNase protection method Winter, E.,Perucho, M., et al., Proc. Natl. Acad. Sci. USA. 82, 7575-7579 (1985)and Myers, R. M., et al., Science, 230, 1242-1246 (1985)! regarding thesequence thereof.

(2) Confirmation of the whole structure of the gene

It has been confirmed that the cDNA obtained by the above procedure hasa novel DNA sequence specified in sequence ID NO 2, and it has beendeduced that the amino acid sequence of a novel protein encoded therebyis as specified in sequence ID NO 1. Proteins each having an amino acidsequence comprising the whole or a part of the sequence specified insequence ID NO 1 have been designated PRLTS proteins by the presentinventors and will be referred to as the same hereinafter. Moreover, thenucleic acid sequence of the genomic DNA has been analyzed and comparedwith that of the cDNA, so that intron-exon combining sites have beenconfirmed and the structures of sequence ID NO's 3, 4, 5, 6, 7, 8 and 9including introns and exons have been elucidated.

(3) Recombinant expression vectors and transformants thereof

The DNA encoding human PRLTS protein obtained by the above-mentionedprocedure or a fragment thereof can be integrated into a suitable vectorand introduced by transfection into a suitable host cell, therebyobtaining a transformant. This transformant can be cultured in aconventional manner. The human PRLTS protein can be produced in largequantity from the culture. More particularly, the DNA encoding the humanPRLTS protein or a fragment thereof can be recombined with a vectorsuitable for expression thereof downstream of the promoter of the vectoraccording to the customary procedure in which a restriction enzyme and aDNA ligase are employed, thereby obtaining a recombinant expressionvector. Although plasmids pBR322 and pUC18 derived from Escherichiacoli, plasmid pUB110 derived from Bacillus subtilis, plasmid pRB15derived from yeast, bacteriophage vectors λgt10 and λgt11, and vectorSV40 may be mentioned as examples of suitable vectors, the vectors arenot particularly limited as long as they can be replicated and amplifiedin the host. With respect to the types of promoter and terminator aswell, they are not particularly limited as long as they are adapted tothe host for use in the expression of the DNA sequence encoding thehuman PRLTS protein and appropriate combinations can be effecteddepending on the host. The employed DNA is by no way limited as long asit encodes the human PRLTS protein. The employed DNA is not limited tothose having the nucleic acid sequences specified in sequence ID NO 2,3, 4, 5, 6, 7, 8 and 9. The DNAs may be those having a nucleic acidsequence resulting from intentional or nonintentional substitution,deletion, insertion and/or addition, conducted independently or incombination, of part of each of the nucleic acid sequences describedabove. The above DNA may be prepared by any nonlimited method, includingbiological and chemical methods.

The thus-obtained recombinant expression vector is introduced into ahost according to any of various methods such as the competent cellmethod J. Mol. Biol., 53, 154 (1970)!, the protoplast method Proc. Natl.Acad. Sci. USA., 75, 1929 (1978)!, the calcium phosphate method Science,221, 551 (1983)!, the in vitro packaging method Proc. Natl. Acad. Sci.USA., 72, 581 (1975)! and the virus vector method Cell, 37, 1053(1984)!, thereby preparing a transformant. For example, Escherichiacoli, Bacillus subtilis, yeast and animal cells can be used as the host.The resultant transformaNt is cultured in a medium suitable for thehost. The culturing is generally effected at 20 to 45° C. and pH 5 to 8.According to necessity, ventilation and agitation are conducted.Separation of the PRLTS protein from the culture and purificationthereof can be effected by a suitable combination of conventionalseparation and purification means. Examples of the above conventionalmeans include salting out, solvent precipitation, dialysis, gelfiltration, electrophoresis, ion exchange chromatography, affinitychromatography and reversed phase high-performance liquidchromatography.

(4) Preparation of antibody

The antibody can be prepared by the use of the whole or a part of thePRLIS protein as the antigen according to the customary procedure. Forexample, a polyclonal antibody is prepared by inoculating an animal suchas a mouse, a guinea-pig and a rabbit by subcutaneous, intramuscular,intraperitoneal or intravenous injection of the antigen more than onetime to thereby satisfactorily immunize the animal, sampling bloodspecimen from the animal and separating sera from the blood specimen.Also, commercially available adjuvants can be used.

The monoclonal antibody can be prepared, for example, by conducting cellfusion of the splenocyte of a mouse immunized with the PRLTS protein anda commercially available mouse myeloma cell to thereby obtain ahybridoma and recovering from the hybridoma culture supernatant or fromthe ascites of a mouse in which the hybridoma has been injected.

The PRLTS protein for use as the antigen does not necessarily have topossess the entire amino acid structure. It may be a peptide having apartial structure thereof, a mutant thereof, a derivative thereof or apeptide resulting from the fusion with another peptide. These may beprepared by any of customary methods including biological and chemicalmethods. The resultant antibody accomplishes identification andquantitative determination of the PRLTS protein in human biospecimensand can be used as diagnostic agents for cancer, carcinoma or tumors.The immunological determination of the PRLTS protein may be achieved inaccordance with publicly known methods, for example, the fluorescentantibody method, the passive agglutination reaction method and theenzyme antibody method.

(5) Gene analysis with respect to human tumor tissue

For example, human blood, body fluid and secretory fluid as well ashuman normal tissues and various human tumor tissues can be used as thebiospecimens subjected to gene analysis. The extraction and preparationof DNA is performed by, for example, the method of Sato et al. Sato, T.,et al., Cancer Res., 50, 7184 (1990)!.

The presence or absence of mutation in the gene can be analyzed with theuse of restriction enzyme fragments of the DNA encoding the human PRLTSprotein according to the present invention as the probe or with the useof an oligonucleotide synthesized by appropriately selecting a nucleicacid sequence located in a suitable position from the above DNA as theprimer.

Further, any defects such as insertion and deletion in the gene of eachspecimen can be detected by the above analyses.

The selected nucleic acid sequence site can be any of the gene's exons,gene's introns and combining sites thereof. Further, artificiallymodified nucleic acid sequences can naturally be employed, by which thecorresponding gene mutations can be detected.

The above analysis can be conducted by, for example, a method in whichamplification of a partial sequence is conducted with the use of primershaving two types of sequences selected according to the PCR method andthe nucleic acid sequence of an amplification product is directlyanalyzed or a method in which this amplification product is integratedinto a plasmid in the same manner as described above, a host cell istransformed by the plasmid and cultured, and the nucleic acid sequenceof the obtained clone is analyzed. Alternatively, the presence orabsence of a specified mutation in the above gene of each specimen canbe directly detected by the use of the ligase chain reaction method Wuet al., Genomics, 4, 560-569 (1989)! and, further, themutant-sequence-specific PCR method Ruano and Kidd, Nucleic AcidResearch, 17, 8392 (1989) and C. R. Newton et al., Nucleic AcidResearch, 17, 2503-2517 (1989)!.

Similarly, a point mutation can be detected with the use of a probeincluding a selected DNA sequence or RNA sequence derived therefromaccording to the SSCP method and the RNase-protection method. Moreover,any mutation in the gene of each specimen in the Southern hybridizationmethod and any abnormality in the quantity of expression attained by thegene of each specimen in the Northern hybridization method can bedetected with the use of such a probe.

The Escherichia coli 68cDNA into which the plasmid containing the DNAencoding the PRLTS protein was introduced was deposited with theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry under the accession number FERM BP-4658 on Apr. 27, 1994.

Expectations are entertained of the use of the human PRLTS protein andthe DNA containing the whole or a part of the DNA encoding this proteinaccording to the present invention as agents for investigating,testing/diagnosing and treating tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows partial deletions in the short arm of the human chromosome8 in human hepatocellular carcinoma (HCC) and lung cancer (LC), in whicheach solid circle refers to the loss of heterozygosity (LOH), while eachopen circle to the retention thereof, and each rectangular frame to thecommon deletion region, and in which each clone is indicated by itsnumber only.

FIG. 2 shows partial deletions in the short arm of the human chromosome8 in colorectal cancer (CRC), in which each solid circle refers to theloss of heterozygosity (LOH), while each open circle to the retentionthereof, and the rectangular frame to the common deletion region, and inwhich each clone is indicated by its number only.

FIG. 3 shows the markers positioned in the common deletion region of theshort arm of the human chromosome 8 and the positions of two YAC clonespresent in the region, in which each marker is indicated by the clonenumber only.

FIG. 4 shows the size of the restriction enzyme fragment identified bythe genomic Southern blotting analyses, with the use of each DNA markerpositioned around the localized common deletion region as the probe, inwhich the size is expressed in kb and each clone is indicated by itsnumber only.

FIG. 5 shows Southern blotting detections of a gene rearrangement inlung cancer, in which N and T represent the DNAs of normal and tumortissues, respectively.

EXAMPLES

The present invention will now be described in greater detail and morespecifically with reference to the following Examples, which by no meanslimit the invention.

Example 1

Isolation of cosmid clones specific to the human chromosome 8 andpreparation of chromosome map

Cosmid clones specific to the human chromosome 8 were isolated inaccordance with the method of Tokino et al. Tokino et al., Am. J. Hum.Genet., 48, 258-268 (1991)!. Specifically, the genomic DNA ofhuman-mouse hybrid cell line A9neo8/t containing a single humanchromosome 8 in a mouse genomic background Koi et al., Jpn. J. CancerRes., 80, 413-418 (1989)! was appropriately digested with restrictionenzyme Sau3AI, and the terminals of the resultant fragments were treatedby partial filling-in in which dATP and dGTP were employed. Then,fragments having a size of 35 to 42 kb were fractionated from thetreated fragments and inserted into cosmid vectors pWEX15 which had beensubjected to digestion with restriction enzyme XhoI and similartreatment by partial filling-in in which dCTP and dTTP were employed.Cosmid clones specific to the human chromosome 8 were isolated byscreening clones containing human DNA fragments from the obtained cosmidclones in accordance with the colony hybridization method in which ³²P-labeled human genomic DNA was used as the probe.

The positions on the chromosome where the above DNA fragments of cosmidclones were to be hybridized thereto were determined in accordance withthe FISH method Inazawa et al., Genomics, 10, 1075-1078 (1991)!.

With respect to the cosmid clones whose positions on the chromosome weredetermined (cosmid markers), the detectability of RFLP was tested inaccordance with the known method Nakamura et al., Am, J. Hum. Genet.,43, 854-859 (1988)!. The restriction enzyme for use was selected fromMspI, TaqI, BglII, PstI, PvuII, RsaI and EcoRI.

Example 2

Determination of the order of cosmid marker sites on the short arm ofthe human chromosome 8

For further limiting the region (8p21 to 8p23) on the short arm of thehuman chromosome 8 which would be commonly deleted in hepatocellularcarcinoma, colorectal cancer and pulmonary nonsmall cell carcinoma andwhich was discovered by the present inventors Emi, M., et al., CancerRes., 52, 5368-5372 (1992)!, the order of 14 polymorphic cosmid markers(RFLP markers) present within the above region which were obtained inExample 1 was determined (see Table 1). Of them, the order of four sites(CI8-190, CI8-245, CI8-487 and MSR-32) had already been determined bythe genetic linkage analysis effected by the present inventors Emi, M.,et al., Genomics, 15, 530-534 (1993)!, and the genetic distance betweenCI8-190 and MSR-32 sites was estimated to be 10 cM. With the use ofindividual cosmid markers including these four markers in combination,the multicolor FISH method Inazawa et al., Jpn. J. Cancer Res., 20,1248-1252 (1992) and Inazawa et al., Cytogenet. Cell Genet., 65, 130-135(1994)! was repeatedly followed, thereby analyzing the relativepositional relationship of the above markers. As a result, the order ofthe above 14 RFLP markers (denoted only by the number with omission of"CI8-") was determined as centromere--1308-1013-190-(1312, 2439)-(245,487)-1051-(2644, MSR35, MSR32)-2014-2195-1344--telomere, although, withrespect to the parenthesized markers, relative position divisions couldnot be attained by this method.

Example 3

Localization of the region of the short arm of the human chromosome 8commonly deleted in hepatocellular carcinoma, pulmonary nonsmall cellcarcinoma and colorectal cancer

Tumor tissues were obtained from operative materials consisting of 102cases of hepatocellular carcinoma, 53 cases of pulmonary nonsmall cellcarcinoma and 123 cases of colorectal cancer. The corresponding normaltissues or peripheral blood samples were obtained from the individualpatients. DNAs were extracted from these tissues and blood samples inaccordance with the known method Sato, T., et al. Cancer Res., 50,7184-7189 (1990)!. Each of the DNAs was digested with a suitablerestriction enzyme, followed by electrophoresis on 1.0% agarose gel, andSouthern-transferred with 0.1 N NaOH/0.1 M NaCl by means of a nylonmembrane Sato, T., et al. Cancer Res., 50, 7184-7189 (1990)!.

On the resultant membranes, Southern hybridization was conducted withthe use of 14 RFLP markers whose order was determined in Example 2 asthe probe Sato, T., et al. Cancer Res., 50, 7184-7189 (1990)!, therebytesting the LOH (loss of heterozygosity) (see Table 1).

In hepatocellular carcinoma, the heterozygosity could be discriminated(informative) by at least one marker in 93 cases, of which in 34 cases(37%) the LOH was detected by at least one marker. In pulmonary nonsmallcell carcinoma, 56 cases were informative with at least one marker, ofwhich in 26 cases (46%) the LOH was detected by at least one marker. Incolorectal cancer, 115 cases were informative with at least one marker,of which in 64 cases (54%) the LOH was detected by at least one marker.

Cases in which the presence of a partial deletion on the short arm ofthe human chromosome 8 (8p) could positively be judged from the aboveresults, i.e., not only could the heterozygosity be discriminated(informative) with at least two markers but also the presence of adeletion portion (LOH detected) and a retention portion (LOH notdetected) was demonstrated were collected and analyzed.

As a result, the common deletion region was localized between twomarkers CI8-245 and CI8-2644 in 11 cases of hepatocellular carcinoma(HCC) (see FIG. 1). Similarly, the common deletion region was localizedbetween two markers CI8-1051 and CI8-2644 in 12 cases of pulmonarynonsmall cell carcinoma (LC) (see FIG. 1). Further, the common deletionregion was localized between two markers CI8-245 and CI8-2644 in 25cases of colorectal cancer (CRC) (see FIG. 2). The deletion region wascommon in these three tissue carcinomas (cancers) and localized in anespecially narrow zone in pulmonary nonsmall cell carcinoma.

Example 4

Preparation of physical map by pulsed field gel electrophoresis (PFGE)

With respect to the common deletion region localized in Example 3, aphysical map was prepared by PFGE analysis in which use was made of 12DNA markers of the above region. Of these, eight DNA markers werepolymorphic cosmid clones employed in the examination of LOH. As for theother four nonpolymorphic markers (CI8-1195, CI8-2429, CI8-2003 andCI8-1372), their positions relative to CI8-1312 and cMSR-32 weredetermined in accordance with the multicolor FISH method.

The genomic DNA was digested with seven types of restriction enzymeshaving rare breakage points, the thus-obtained fragments were subjectedto PFGE, and a Southern blotting analysis was conducted with the use ofeach DNA marker as the probe. The size of the genomic DNA fragmentidentified by each of 12 DNA markers was summarized in FIG. 4.

No gap was recognized on this PFGE map, and the order of all the markersexcept cMSR-32 and CI8-2429 became apparent. The size of the mostnarrowly localized common deletion region (between CI8-1051 andCI8-2644) recognized in pulmonary nonsmall cell carcinoma was estimatedto be about 600 kb.

Example 5

Preparation of the cosmid contig of the common deletion region

In order to isolate the genes present in this 600 kb region, the presentinventors isolated two YAC clones, i.e., Y812 and Y738, each containinga genomic region ranging from cosmid marker CI8-487 to cosmid markerCI8-2003 (see FIG. 3). A cosmid library was prepared from the DNAs ofthe above two YAC clones, and 74 clones having human DNAs were obtained.A cosmid contig map of these 74 cosmid clones was constructed inaccordance with the Southern hybridization method.

Example 6

Isolation of genes from the common deletion region

34 cosmid clones covering the whole of the common deletion region wereselected from the cosmid contig obtained in Example 5, and sequencescapable of serving as exons were searched in accordance with the exonamplification method Buckler et al., Proc. Natl. Acad. Sci. USA., 88,4005-4009 (1991)!. As a result, 54 exon-like DNA fragments wereobtained. cDNA libraries derived from a fetal lung and a fetal brainwere each screened with the use of the above DNA fragments as probes.Thus, six mutually different cDNA clones were obtained.

Example 7

Detection of the gene rearrangement in tumor tissue

The six types of cDNA clones were individually used as the probe toinvestigate whether any tumor-tissue-specific gene rearrangement wouldbe detected. In particular, Southern blotting analysis was conductedwith the use of the DNAs of the above cDNA clones as probes forfragments obtained by digestion of the DNAs of tumor tissues and normaltissues corresponding thereto with restriction enzyme(s) (EcoRI andHindIII, PvuII or PstI) to thereby detect any major abnormality instructural genes such as deletion, duplication, amplification andtranslocation occurring in tumor cells, namely, any gene rearrangement.A panel of DNAs in 295 cases consisting of 102 cases of hepatocellularcarcinoma, 70 cases of pulmonary nonsmall cell carcinoma and 123 casesof colorectal cancer was investigated. As a result, when one cDNA clonewas used as the probe, a gene rearrangement was detected in one case ofpulmonary nonsmall cell carcinoma (see FIG. 5). Comparison in Southernblotting patterns of DNAs between the tumor tissue and the normal tissueshowed the occurrence of this gene reconstitution in atumor-tissue-specific manner.

It was confirmed that this clone was derived from the above commondeletion region because it contained a sequence derived from cosmidcCI8-3068 whose position was in the middle between cCI8-1051 andcCI-2644 as found by the multicolor FISH. In this tumor, it was apparentthat "two hit" somatic mutation occurred at the site including the abovecDNA because the LOH was recognized with respect to both outside markerscCI8-245 (D8S335) and cMSR-32 (MSR).

Example 8

Structure of cDNA

This cDNA clone was isolated from the fetal lung cDNA library, and itwas confirmed that this cDNA clone consisted of 1502 bp and had a novelDNA sequence including a 61 bp 5'-noncoding region, 1128 bp codingregion and 313 bp 3'-noncoding region (see Sequence ID NO 2). This cDNAsequence contained an open reading frame which encoded a novel proteincomposed of 375 amino acids (PRLTS protein, see Sequence ID NO 1).

Example 9

Determination of the structure of genomic DNA

The presence of seven exons was confirmed by comparing the sequence ofthe genomic DNA of the cosmid clone with that of the cDNA obtained inExample 8 with respect to the sequence of the genomic DNA around exons.The exon-containing genomic DNA sequences are set forth in Sequence IDNO's 3, 4, 5, 6, 7, 8 and 9, in which the amino acid number correspondsto that of Sequence ID NO 1 to thereby represent the position number inthe PRLTS protein.

Example 10

Detection of the gene mutation in tumor tissue

Single-strand conformation polymorphism (SSCP) analysis Orita, M., etal., Genomics, 5, 874-879 (1984) and Orita, M., et al., Cell, 60,509-520 (1990)! was conducted on the DNAs of 48 cases of hepatocellularcarcinoma, 31 cases of pulmonary nonsmall cell carcinoma and 28 cases ofcolorectal cancer, thereby testing mutations in the genes. The SSCPanalysis was conducted by amplifying the individual exons with the useof PCR primers having sequences (Sequence ID NO's 10 and 11, Sequence IDNO's 12 and 13, Sequence ID NO's 14 and 15, Sequence ID NO's 16 and 17,Sequence ID NO's 18 and 19, and Sequence ID NO's 20 and 21) designedfrom the sequence of the genomic DNA obtained in Example 9. The nucleicacid sequences of Sequence ID's NO 10 to 21 were designed from thesequences of Sequence ID's NO 3 to 9. There are the followingrelationships:

Sequence ID NO 10=Sequence ID NO 4, Nos. 3-24,

Sequence ID NO 11=Sequence ID NO 4, Nos. 281-300 antisense,

Sequence ID NO 12=Sequence ID NO 5, Nos. 49-71,

Sequence ID NO 13=Sequence ID NO 5, Nos. 393-415 antisense,

Sequence ID NO 14=Sequence ID NO 6, Nos. 5-24,

Sequence ID NO 15=Sequence ID NO 6, Nos. 207-231 antisense,

Sequence ID NO 16=Sequence ID NO 7, Nos. 34-53,

sequence ID NO 17=Sequence ID NO 7, Nos. 372-393 antisense,

Sequence ID NO 18=Sequence ID NO 8, Nos. 5-24,

Sequence ID NO 19=Sequence ID NO 8, Nos. 191-211 antisense,

Sequence ID NO 20=Sequence ID NO 9, Nos. 21-41, and

Sequence ID NO 21=Sequence ID NO 9, Nos. 263-285 antisense.

PCR products which had changes recognized in electrophoresis patterns asa result of the SSCP analysis were cloned and their nucleic acidsequences were determined. Consequently, point mutations accompanied byamino acid substitution were found in one case of colorectal cancer(CRC) and one case of hepatocellular carcinoma (HCC) and a mutationaccompanied by 2-base deletion in one case of hepatocellular carcinoma(HCC) (see Table 2). By comparison of the nucleic acid sequence of thetumor tissue with that of the corresponding normal tissue, it wasconfirmed that these mutations were present only in tumor tissues.

                  TABLE 2    ______________________________________    tumor    codon      mutation       LOH    ______________________________________    CRC20    23         CAC (His) to TAC (Tyr)                                       +    HCC74    302        GCG (Val) to GTG (Ala)                                       +    HCC107   175        CTTTG to CTG   +    ______________________________________

In one case of colorectal cancer, CRC 20, a point mutation occurred fromC to T, so that codon 23 changed from histidine to tyrosine. In thiscase, it was demonstrated that no normal C-containing sequence wasdetected in the nucleic acid sequence of the tumor DNA and that allelicdeletion occurred. In hepatocellular carcinoma HCC 74, a point mutationoccurred from C to T, so that codon 302 changed from valine to alanine.In another case of hepatocellular carcinoma, HCC 107, a frame shiftoccurred because of the mutation brought about by deletion of two basesat codon 175, so that a stop codon was formed downstream. In these casesas well, allelic deletion occurred in the tumor cell.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:  21    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  375              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  protein    -     (vi) ORIGINAL SOURCE:    #sapiens  (A) ORGANISM:  Homo    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: human feta - #l lung cDNA library    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 1:    - Met Lys Val Trp Leu Leu Leu Gly Leu Leu Le - #u Val His Glu Ala Leu    #                 15    - Glu Asp Val Thr Gly Gln His Leu Pro Lys As - #n Lys Arg Pro Lys Glu    #             30    - Pro Gly Glu Asn Arg Ile Lys Pro Thr Asn Ly - #s Lys Val Lys Pro Lys    #        45    - Ile Pro Lys Met Lys Asp Arg Asp Ser Ala As - #n Ser Ala Pro Lys Thr    #    60    - Gln Ser Ile Met Met Gln Val Leu Asp Lys Gl - #y Arg Phe Gln Lys Pro    #80    - Ala Ala Thr Leu Ser Leu Leu Ala Gly Gln Th - #r Val Glu Leu Arg Cys    #                95    - Lys Gly Ser Arg Ile Gly Trp Ser Tyr Pro Al - #a Tyr Leu Asp Thr Phe    #           110    - Lys Asp Ser Arg Leu Ser Val Lys Gln Asn Gl - #u Arg Tyr Gly Gln Leu    #       125    - Thr Leu Val Asn Ser Thr Ser Ala Asp Thr Gl - #y Glu Phe Ser Cys Trp    #   140    - Val Gln Leu Cys Ser Gly Tyr Ile Cys Arg Ly - #s Asp Glu Ala Lys Thr    145                 1 - #50                 1 - #55                 1 -    #60    - Gly Ser Thr Tyr Ile Phe Phe Thr Glu Lys Gl - #y Glu Leu Phe Val Pro    #               175    - Ser Pro Ser Tyr Phe Asp Val Val Tyr Leu As - #n Pro Asp Arg Gln Ala    #           190    - Val Val Pro Cys Arg Val Thr Val Leu Ser Al - #a Lys Val Thr Leu His    #       205    - Arg Glu Phe Pro Ala Lys Glu Ile Pro Ala As - #n Gly Thr Asp Ile Val    #   220    - Tyr Asp Met Lys Arg Gly Phe Val Tyr Leu Gl - #n Pro His Ser Glu His    225                 2 - #30                 2 - #35                 2 -    #40    - Gln Gly Val Val Tyr Cys Arg Ala Glu Ala Gl - #y Gly Arg Ser Gln Ile    #               255    - Ser Val Lys Tyr Gln Leu Leu Tyr Val Ala Va - #l Pro Ser Gly Pro Pro    #           270    - Ser Thr Thr Ile Leu Ala Ser Ser Asn Lys Va - #l Lys Ser Gly Asp Asp    #       285    - Ile Ser Val Leu Cys Thr Val Leu Gly Glu Pr - #o Asp Val Glu Val Glu    #   300    - Phe Thr Trp Ile Phe Pro Gly Gln Lys Asp Gl - #u Arg Pro Val Thr Ile    305                 3 - #10                 3 - #15                 3 -    #20    - Gln Asp Thr Trp Arg Leu Ile His Arg Gly Le - #u Gly His Thr Thr Arg    #               335    - Ile Ser Gln Ser Val Ile Thr Val Glu Asp Ph - #e Glu Thr Ile Asp Ala    #           350    - Gly Tyr Tyr Ile Cys Thr Ala Gln Asn Leu Gl - #n Gly Gln Thr Thr Val    #       365    - Ala Thr Thr Val Glu Phe Ser    #   375    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1502              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  cDNA to mRNA    -     (vi) ORIGINAL SOURCE:    #sapiens  (A) ORGANISM:  Homo    -    (vii) IMMEDIATE SOURCE:    #fetal lung cDNA libraryhuman    -     (ix) FEATURE:              (A) NAME/KEY:  CDS              (B) LOCATION:  62..1189              (C) IDENTIFICATION METHOD: - # experimental examination    #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - CCTGCGTCCC CGCCCCGCNC AGCCGCCGCG CTCCTGCNCT CCGAGGTCCG AG - #GTTCCCGA      60    #CTG GTG CAC GAA GCG        106 GGT CTT CTG      Met Lys Val Trp Leu Leu Leu Gly Leu L - #eu Leu Val His Glu Ala    # 15    - CTG GAG GAT GTT ACT GGC CAA CAC CTT CCC AA - #G AAC AAG CGT CCA AAA     154    Leu Glu Asp Val Thr Gly Gln His Leu Pro Ly - #s Asn Lys Arg Pro Lys    #                 30    - GAA CCA GGA GAG AAT AGA ATC AAA CCT ACC AA - #C AAG AAG GTG AAG CCC     202    Glu Pro Gly Glu Asn Arg Ile Lys Pro Thr As - #n Lys Lys Val Lys Pro    #             45    - AAA ATT CCT AAA ATG AAG GAC AGG GAC TCA GC - #C AAT TCA GCA CCA AAG     250    Lys Ile Pro Lys Met Lys Asp Arg Asp Ser Al - #a Asn Ser Ala Pro Lys    #         60    - ACG CAG TCT ATC ATG ATG CAA GTG CTG GAT AA - #A GGT CGC TTC CAG AAA     298    Thr Gln Ser Ile Met Met Gln Val Leu Asp Ly - #s Gly Arg Phe Gln Lys    #     75    - CCC GCC GCT ACC CTG AGT CTG CTG GCG GGG CA - #A ACT GTA GAG CTT CGA     346    Pro Ala Ala Thr Leu Ser Leu Leu Ala Gly Gl - #n Thr Val Glu Leu Arg    # 95    - TGT AAA GGG AGT AGA ATT GGG TGG AGC TAC CC - #T GCG TAT CTG GAC ACC     394    Cys Lys Gly Ser Arg Ile Gly Trp Ser Tyr Pr - #o Ala Tyr Leu Asp Thr    #               110    - TTT AAG GAT TCT CGC CTC AGC GTC AAG CAG AA - #T GAG CGC TAC GGC CAG     442    Phe Lys Asp Ser Arg Leu Ser Val Lys Gln As - #n Glu Arg Tyr Gly Gln    #           125    - TTG ACT CTG GTC AAC TCC ACC TCG GCA GAC AC - #A GGT GAA TTC AGC TGC     490    Leu Thr Leu Val Asn Ser Thr Ser Ala Asp Th - #r Gly Glu Phe Ser Cys    #       140    - TGG GTG CAG CTC TGC AGC GGC TAC ATC TGC AG - #G AAG GAC GAG GCC AAA     538    Trp Val Gln Leu Cys Ser Gly Tyr Ile Cys Ar - #g Lys Asp Glu Ala Lys    #   155    - ACG GGC TCC ACC TAC ATC TTT TTT ACA GAG AA - #A GGA GAA CTC TTT GTA     586    Thr Gly Ser Thr Tyr Ile Phe Phe Thr Glu Ly - #s Gly Glu Leu Phe Val    160                 1 - #65                 1 - #70                 1 -    #75    - CCT TCT CCC AGC TAC TTC GAT GTT GTC TAC TT - #G AAC CCG GAC AGA CAG     634    Pro Ser Pro Ser Tyr Phe Asp Val Val Tyr Le - #u Asn Pro Asp Arg Gln    #               190    - GCT GTG GTT CCT TGT CGG GTG ACC GTG CTG TC - #G GCC AAA GTC ACG CTC     682    Ala Val Val Pro Cys Arg Val Thr Val Leu Se - #r Ala Lys Val Thr Leu    #           205    - CAC AGG GAA TTC CCA GCC AAG GAG ATC CCA GC - #C AAT GGA ACG GAC ATT     730    His Arg Glu Phe Pro Ala Lys Glu Ile Pro Al - #a Asn Gly Thr Asp Ile    #       220    - GTT TAT GAC ATG AAG CGG GGC TTT GTG TAT CT - #G CAA CCT CAT TCC GAG     778    Val Tyr Asp Met Lys Arg Gly Phe Val Tyr Le - #u Gln Pro His Ser Glu    #   235    - CAC CAG GGT GTG GTT TAC TGC AGG GCG GAG GC - #C GGG GGC AGA TCT CAG     826    His Gln Gly Val Val Tyr Cys Arg Ala Glu Al - #a Gly Gly Arg Ser Gln    240                 2 - #45                 2 - #50                 2 -    #55    - ATC TCC GTC AAG TAC CAG CTG CTC TAC GTG GC - #G GTT CCC AGT GGC CCT     874    Ile Ser Val Lys Tyr Gln Leu Leu Tyr Val Al - #a Val Pro Ser Gly Pro    #               270    - CCC TCA ACA ACC ATC TTG GCT TCT TCA AAC AA - #A GTG AAA AGT GGG GAC     922    Pro Ser Thr Thr Ile Leu Ala Ser Ser Asn Ly - #s Val Lys Ser Gly Asp    #           285    - GAC ATC AGT GTG CTC TGC ACT GTC CTG GGG GA - #G CCC GAT GTG GAG GTG     970    Asp Ile Ser Val Leu Cys Thr Val Leu Gly Gl - #u Pro Asp Val Glu Val    #       300    - GAG TTC ACC TGG ATC TTC CCA GGG CAG AAG GA - #T GAA AGG CCT GTG ACG    1018    Glu Phe Thr Trp Ile Phe Pro Gly Gln Lys As - #p Glu Arg Pro Val Thr    #   315    - ATC CAA GAC ACT TGG AGG TTG ATC CAC AGA GG - #A CTG GGA CAC ACC ACG    1066    Ile Gln Asp Thr Trp Arg Leu Ile His Arg Gl - #y Leu Gly His Thr Thr    320                 3 - #25                 3 - #30                 3 -    #35    - AGA ATC TCC CAG AGT GTC ATT ACA GTG GAA GA - #C TTC GAG ACG ATT GAT    1114    Arg Ile Ser Gln Ser Val Ile Thr Val Glu As - #p Phe Glu Thr Ile Asp    #               350    - GCA GGA TAT TAC ATT TGC ACT GCT CAG AAT CT - #T CAA GGA CAG ACC ACA    1162    Ala Gly Tyr Tyr Ile Cys Thr Ala Gln Asn Le - #u Gln Gly Gln Thr Thr    #           365    - GTA GCT ACC ACT GTT GAG TTT TCC TGACTTGGAA AA - #GGAAATGT AATGAACTTA    1216    Val Ala Thr Thr Val Glu Phe Ser    #       375    - TGGAAAGCCC ATTTGTGTAC ACAGTCAGCT TTGGGGTTCC TTTTATTAGT GC - #TTTGCCAG    1276    - AGGCTGATGT CAAGCACCAC ACCCCAACCC CAGCGTCTCG TGAGTCCGAC CC - #AGACATCC    1336    - AAACTAAAAG GAAGTCATCC AGTCTATTCA CAGAAGTGTT AACTTTTCTA AC - #AGAAAGCA    1396    - TGATTTTGAT TGCTTACCTA CATACGTGTT CCTAGTTTTT ATACATGTGT AA - #ACAATTTT    1456    #               1502TAT TAAATGAGCA CGTTTTTGTA AAAAAT    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  281              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  genomic DNA    -     (vi) ORIGINAL SOURCE:    #sapiens  (A) ORGANISM:  Homo    -    (vii) IMMEDIATE SOURCE:    #cosmid libraryIBRARY: human DNA    -     (ix) FEATURE:    #1        (A) NAME/KEY:  exon              (B) LOCATION:  99..150              (C) IDENTIFICATION METHOD: - #  experimental examination    #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - AAATTCCCCA ACTTTTTCCC CCAAACCTTG TTCCTCCTGA AGAAACCGAA TC - #CTCCCGCT      60    - TCGGCGTCCC AGGAGCCCGC CCCTCGCCCG CCGCCTCCCC TGCGTCCCCG CC - #CCGCNCAG     120    - CCGCCGCGCT CCTGCNCTCC GAGGTCCGAG GTTCCCGAGA TNAAGGTCTG GC - #TGCTGCTT     180    - GGTCTTCTGC TGGTGCNCCA AGCGATGGAG GATGGTGAGT GACTCTGGGC GC - #GGGGCCAC     240    #  281             ACTT TAGCCGGGAC CCGAAGTTTT T    - (2) INFORMATION FOR SEQ ID NO: 4:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  323              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #ld              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  genomic DNA    -     (vi) ORIGINAL SOURCE:    #sapiens  (A) ORGANISM:  Homo    -    (vii) IMMEDIATE SOURCE:    #cosmid libraryIBRARY: human DNA    -     (ix) FEATURE:    #2        (A) NAME/KEY:  exon              (B) LOCATION:  128..191              (C) IDENTIFICATION METHOD: - #  experimental examination    #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - AAACCTTGTT CCTCCTGAAG AAACCGAATC CTCCCGCTTC GGCGTCCCAG GA - #GCCCGCCC      60    - CTCGCCCGCC GCCTCCCCTG CGTCCCCGCC CCGCGCAGCC GCCGGCTCCT GC - #GCTCCGAG     120    #GGT CTT CTG CTG GTG    172 GTC TGG CTG CTG CTT    #Met Lys Val Trp Leu Leu Leu Gly Leu Leu L - #eu Val    #                 10    #GGCCACCTAG           221 G GTGAGTGACT CTGGGCGCGG    His Glu Ala Leu Glu Asp Val             15    - CTTGTGCCCT GACTTTAGCC GGGACCCGAA GCCCCCGCCG CCCTCCTGCC AG - #CTCTTGGT     281    # 323              GGTG GCTCAGCCCC CGCNCCACTG CC    - (2) INFORMATION FOR SEQ ID NO: 5:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  486              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  genomic DNA    -     (vi) ORIGINAL SOURCE:    #sapiens  (A) ORGANISM:  Homo    -    (vii) IMMEDIATE SOURCE:    #cosmid libraryIBRARY: human DNA    -     (ix) FEATURE:    #3        (A) NAME/KEY:  exon              (B) LOCATION:  74..371              (C) IDENTIFICATION METHOD: - #  experimental examination    #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - AAAAAGTTAT TGGCCTCAAA TATTCCAAAA ATGTCATTAC TACAGNGNAT TT - #CTCTCTCC      60    - TTACGTTTTG CAG TT ACT GGC CAA CAC CTT CCC AAG - # AAC AAG CGT CCA AAA     111                  Val Thr Gl - #y Gln His Leu Pro Lys Asn Lys Arg Pro Lys    # 30    - GAA CCA GGA GAG AAT AGA ATC AAA CCT ACC AA - #C AAG AAG GTG AAG CCC     159    Glu Pro Gly Glu Asn Arg Ile Lys Pro Thr As - #n Lys Lys Val Lys Pro    #             45    - AAA ATT CCT AAA ATG AAG GAC AGG GAC TCA GC - #C AAT TCA GCA CCA AAG     207    Lys Ile Pro Lys Met Lys Asp Arg Asp Ser Al - #a Asn Ser Ala Pro Lys    #         60    - ACG CAG TCT ATC ATG ATG CAA GTG CTG GAT AA - #A GGT CGC TTC CAG AAA     255    Thr Gln Ser Ile Met Met Gln Val Leu Asp Ly - #s Gly Arg Phe Gln Lys    #     75    - CCC GCC GCT ACC CTG AGT CTG CTG GCG GGG CA - #A ACT GTA GAG CTT CGA     303    Pro Ala Ala Thr Leu Ser Leu Leu Ala Gly Gl - #n Thr Val Glu Leu Arg    # 95    - TGT AAA GGG AGT AGA ATT GGG TGG AGC TAC CC - #T GCG TAT CTG GAC ACC     351    Cys Lys Gly Ser Arg Ile Gly Trp Ser Tyr Pr - #o Ala Tyr Leu Asp Thr    #               110    - TTT AAG GAT TCT CGC CTC AG GTAANCATTT TTTTTTAAAN - # CTGTGTAGGG     401    Phe Lys Asp Ser Arg Leu Ser                115    - TTGAGGATTT GTAATAGTTC AAAATTCCTT CTTAACTATT ATTACACATT GT - #TTCTGACA     461    #              486 AATA GATCA    - (2) INFORMATION FOR SEQ ID NO: 6:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  231              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  genomic DNA    -     (vi) ORIGINAL SOURCE:    #sapiens  (A) ORGANISM:  Homo    -    (vii) IMMEDIATE SOURCE:    #cosmid libraryIBRARY: human DNA    -     (ix) FEATURE:    #4        (A) NAME/KEY:  exon              (B) LOCATION:  53..204              (C) IDENTIFICATION METHOD: - #  experimental examination    #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - GGGCCCCAGC CAGGTCTGAT TTGCTTTGAG TTCATGTGTC TNTTATTCCT NG - # C GTC      56    #  Ser Val    - AAG CAG AAT GAG CGC TAC GGC CAG TTG ACT CT - #G GTC AAC TCC ACC TCG     104    Lys Gln Asn Glu Arg Tyr Gly Gln Leu Thr Le - #u Val Asn Ser Thr Ser    120                 1 - #25                 1 - #30                 1 -    #35    - GCA GAC ACA GGT GAA TTC AGC TGC TGG GTG CA - #G CTC TGC AGC GGC TAC     152    Ala Asp Thr Gly Glu Phe Ser Cys Trp Val Gl - #n Leu Cys Ser Gly Tyr    #               150    - ATC TGC AGG AAG GAC GAG GCC AAA ACG GGC TC - #C ACC TAC ATC TTT TTT     200    Ile Cys Arg Lys Asp Glu Ala Lys Thr Gly Se - #r Thr Tyr Ile Phe Phe    #           165    #         231      TGGTGCATTA ATGGAAC    Thr Glu    - (2) INFORMATION FOR SEQ ID NO: 7:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  423              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  genomic DNA    -     (vi) ORIGINAL SOURCE:    #sapiens  (A) ORGANISM:  Homo    -    (vii) IMMEDIATE SOURCE:    #cosmid libraryIBRARY: human DNA    -     (ix) FEATURE:    #5        (A) NAME/KEY:  exon              (B) LOCATION:  61..354              (C) IDENTIFICATION METHOD: - #  experimental examination    #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - GNGTGCTNTN ACTTTGCGTC TCCGGAGTGN ANAAGCCTGT GCTCTTCCTT CC - #CTTNGCAG      60    - AG AAA GGA GAA CTC TTT GTA CCT TCT CCC AGC - # TAC TTC GAT GTT GTC     107    #Tyr Phe Asp Val Valhe Val Pro Ser Pro Ser    #  180    - TAC TTG AAC CCG GAC AGA CAG GCT GTG GTT CC - #T TGT CGG GTG ACC GTG     155    Tyr Leu Asn Pro Asp Arg Gln Ala Val Val Pr - #o Cys Arg Val Thr Val    185                 1 - #90                 1 - #95                 2 -    #00    - CTG TCG GCC AAA GTC ACG CTC CAC AGG GAA TT - #C CCA GCC AAG GAG ATC     203    Leu Ser Ala Lys Val Thr Leu His Arg Glu Ph - #e Pro Ala Lys Glu Ile    #               215    - CCA GCC AAT GGA ACG GAC ATT GTT TAT GAC AT - #G AAG CGG GGC TTT GTG     251    Pro Ala Asn Gly Thr Asp Ile Val Tyr Asp Me - #t Lys Arg Gly Phe Val    #           230    - TAT CTG CAA CCT CAT TCC GAG CAC CAG GGT GT - #G GTT TAC TGC AGG GCG     299    Tyr Leu Gln Pro His Ser Glu His Gln Gly Va - #l Val Tyr Cys Arg Ala    #       245    - GAG GCC GGG GGC AGA TCT CAG ATC TCC GTC AA - #G TAC CAG CTG CTC TAC     347    Glu Ala Gly Gly Arg Ser Gln Ile Ser Val Ly - #s Tyr Gln Leu Leu Tyr    #   260    #TGGTCAGTTT     404TGG CCACCCCTGC CTAGATTCTA GTTAGTCCCC    Val Ala Val    265    #423               CTG    - (2) INFORMATION FOR SEQ ID NO: 8:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  239              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  genomic DNA    -     (vi) ORIGINAL SOURCE:    #sapiens  (A) ORGANISM:  Homo    -    (vii) IMMEDIATE SOURCE:    #cosmid libraryIBRARY: human DNA    -     (ix) FEATURE:    #6        (A) NAME/KEY:  exon              (B) LOCATION:  46..185              (C) IDENTIFICATION METHOD: - #  experimental examination    #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - TTACACTCGG GGTACTCACT CTGCCTGTTT CGTGCTTGCT TCCAG TT C - #CC AGT      53    #             Val Pro Ser    - GGC CCT CCC TCA ACA ACC ATC TTG GCT TCT TC - #A AAC AAA GTG AAA AGT     101    Gly Pro Pro Ser Thr Thr Ile Leu Ala Ser Se - #r Asn Lys Val Lys Ser    270                 2 - #75                 2 - #80                 2 -    #85    - GGG GAC GAC ATC AGT GTG CTC TGC ACT GTC CT - #G GGG GAG CCC GAT GTG     149    Gly Asp Asp Ile Ser Val Leu Cys Thr Val Le - #u Gly Glu Pro Asp Val    #               300    - GAG GTG GAG TTC ACC TGG ATC TTC CCA GGG CA - #G AAG GTAAGTGTTG     195    Glu Val Glu Phe Thr Trp Ile Phe Pro Gly Gl - #n Lys    #           310    #239               CCTG CGTCTCAGCC TCTGCATCTC AGCC    - (2) INFORMATION FOR SEQ ID NO: 9:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  551              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  genomic DNA    -     (vi) ORIGINAL SOURCE:    #sapiens  (A) ORGANISM:  Homo    -    (vii) IMMEDIATE SOURCE:    #cosmid libraryIBRARY: human DNA    -     (ix) FEATURE:    #7        (A) NAME/KEY:  exon              (B) LOCATION:  50..551              (C) IDENTIFICATION METHOD: - #  experimental examination    #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - ATAGCAGCTT GTCCCTCTTG CTTCAGTCTT TGTGGGTGTC GTTAAACAG GAT - # GAA      55    #Asp Glu    #    315    - AGG CCT GTG ACG ATC CAA GAC ACT TGG AGG TT - #G ATC CAC AGA GGA CTG     103    Arg Pro Val Thr Ile Gln Asp Thr Trp Arg Le - #u Ile His Arg Gly Leu    #               330    - GGA CAC ACC ACG AGA ATC TCC CAG AGT GTC AT - #T ACA GTG GAA GAC TTC     151    Gly His Thr Thr Arg Ile Ser Gln Ser Val Il - #e Thr Val Glu Asp Phe    #           345    - GAG ACG ATT GAT GCA GGA TAT TAC ATT TGC AC - #T GCT CAG AAT CTT CAA     199    Glu Thr Ile Asp Ala Gly Tyr Tyr Ile Cys Th - #r Ala Gln Asn Leu Gln    #       360    - GGA CAG ACC ACA GTA GCT ACC ACT GTT GAG TT - #T TCC TGACTTGGAA     245    Gly Gln Thr Thr Val Ala Thr Thr Val Glu Ph - #e Ser    #   375    - AAGGAAATGT AATGAACTTA TGGAAAGCCC ATTTGTGTAC ACAGTCAGCT TT - #GGGGTTCC     305    - TTTTATTAGT GCTTTGCCAG AGGCTGATGT CAAGCACCAC ACCCCAACCC CA - #GCGTCTCG     365    - TGAGTCCGAC CCAGACATCC AAACTAAAAG GAAGTCATCC AGTCTATTCA CA - #GAAGTGTT     425    - AACTTTTCTA ACAGAAAGCA TGATTTTGAT TGCTTACCTA CATACGTGTT CC - #TAGTTTTT     485    - ATACATGTGT AAACAATTTT ATATAATCAA TCATTTCTAT TAAATGAGCA CG - #TTTTTGTA     545    #          551    - (2) INFORMATION FOR SEQ ID NO: 10:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  22              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #                 22GAA AC    - (2) INFORMATION FOR SEQ ID NO: 11:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  20              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 20               TAGA    - (2) INFORMATION FOR SEQ ID NO: 12:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  23              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #                23GTTT TGC    - (2) INFORMATION FOR SEQ ID NO: 13:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  23              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #                23CTAC ACA    - (2) INFORMATION FOR SEQ ID NO: 14:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  20              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 20               TTGC    - (2) INFORMATION FOR SEQ ID NO: 15:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  25              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #               25 AAGT ATTTT    - (2) INFORMATION FOR SEQ ID NO: 16:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  20              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 20               TCCC    - (2) INFORMATION FOR SEQ ID NO: 17:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  22              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #                 22AGG CA    - (2) INFORMATION FOR SEQ ID NO: 18:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  20              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 20               CTGC    - (2) INFORMATION FOR SEQ ID NO: 19:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  21              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #21                CAAC A    - (2) INFORMATION FOR SEQ ID NO: 20:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  21              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #20:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #21                TGTC G    - (2) INFORMATION FOR SEQ ID NO: 21:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  23              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  other nucleic aci - #d (synthetic DNA)    #21:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #                23TCCA TAA    __________________________________________________________________________

What we claim is:
 1. A method of analyzing a gene which encodes a PRLTSprotein, said method comprising:a) hybridizing a DNA probe to a genewhich encodes a PRLTS protein, said probe comprising at least 6consecutive nucleotides of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 9 or atleast 6 consecutive nucleotides of the full complement of SEQ ID NO: 2,3, 4, 5, 6, 7, 8, or 9; and b) determining whether a mutation hasoccurred in said gene, thereby analyzing a gene which encodes a PRLTSprotein.
 2. A method of analyzing a gene which encodes a PRLTS protein,said method comprising:a) hybridizing a DNA primer to a gene whichencodes a PRLTS protein, said primer comprising at least 6 consecutivenucleotides of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 9 or at least 6consecutive nucleotides of the full complement of SEQ ID NO: 2, 3, 4, 5,6, 7, 8, or 9; and b) determining whether a mutation has occurred insaid gene, thereby analyzing a gene which encodes a PRLTS protein. 3.The method of claim 1 wherein said DNA probe comprises at least 12consecutive nucleotides of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 9 or atleast 12 consecutive nucleotides of the full complement of SEQ ID NO: 2,3, 4, 5, 6, 7, 8, or
 9. 4. The method of claim 2 wherein said DNA primercomprises at least 12 consecutive nucleotides of SEQ ID NO: 2, 3, 4, 5,6, 7, 8, or 9 or at least 12 consecutive nucleotides of the fullcomplement of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or
 9. 5. The method ofclaim 1 wherein said DNA probe comprises at least 25 consecutivenucleotides of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 9 or at least 25consecutive nucleotides of the full complement of SEQ ID NO: 2, 3, 4, 5,6, 7, 8, or
 9. 6. The method of claim 2 wherein said DNA primercomprises at least 25 consecutive nucleotides of SEQ ID NO: 2, 3, 4, 5,6, 7, 8, or 9 or at least 25 consecutive nucleotides of the fullcomplement of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or
 9. 7. The method ofclaim 1 wherein said DNA probe consists of SEQ ID NO: 2, 3, 4, 5, 6, 7,8, or 9 or the full complement of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 9.